Liquid Chromatography Mass Spectrometry Detection of Antibiotic Agents in Sputum from Persons with Cystic Fibrosis.

Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at subnanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.

Cystic Fibrosis-Associated Stenotrophomonas maltophilia Strain-Specific Adaptations and Responses to pH.

The airway fluids of cystic fibrosis (CF) patients contain local pH gradients and are more acidic than those of healthy individuals. pH is a critical factor that is often overlooked in studies seeking to recapitulate the infection microenvironment. We sought to determine the impact of pH on the physiology of a ubiqituous yet understudied microbe, Stenotrophomonas maltophilia Phylogenomics was first used to reconstruct evolutionary relationships between 74 strains of S. maltophilia (59 from CF patients). Neither the core genome (2,158 genes) nor the accessory genome (11,978 genes) distinguish the CF and non-CF isolates; however, strains from similar isolation sources grouped into the same subclades. We grew two human and six CF S. maltophilia isolates from different subclades at a range of pH values and observed impaired growth and altered antibiotic tolerances at pH 5. Transcriptomes revealed increased expression of both antibiotic resistance and DNA repair genes in acidic conditions. Although the gene expression profiles of S. maltophilia in lab cultures and CF sputum were distinct, we found that the same genes associated with low pH were also expressed during infection, and the higher pH cultures were more similar to sputum metatranscriptomes. Our findings suggest that S. maltophilia is not well adapted to acidity and may cope with low pH by expressing stress response genes and colonizing less acidic microenvironments. As a whole, our study underlines the impact of microenvironments on bacterial colonization and adaptation in CF infections.IMPORTANCE Understanding bacterial responses to physiological conditions is an important priority for combating opportunistic infections. The majority of CF patients succumb to inflammation and necrosis in the airways, arising from chronic infection due to ineffective mucociliary clearance. Steep pH gradients characterize the CF airways but are not often incorporated in standard microbiology culture conditions. Stenotrophomonas maltophilia is a prevalent CF opportunistic pathogen also found in many disparate environments, yet this bacterium's contribution to CF lung damage and its response to changing environmental factors remain largely understudied. Here, we show that pH impacts the physiology and antibiotic susceptibility of S. maltophilia, with implications for the development of relevant in vitro models and assessment of antibiotic sensitivity.

Getting Our Fingers on the Pulse of Slow-Growing Bacteria in Hard-To-Reach Places.

Chronic infections with slow-growing pathogens have plagued humans throughout history. However, assessing the identities and growth rates of bacteria in an infection has remained an elusive goal. Neubauer et al. (J. Bacteriol. 200:e00365-18, 2018, https://doi.org/10.1128/JB.00365-18) combine two cutting-edge approaches to make progress on both fronts: probing specific RNA molecules to assess the identity of actively transcribing microbes and measuring growth rates through incorporation of stable isotope labels. They found that growth rates of pathogens were relatively stable during antibacterial therapy. The article delves into a basic and unanswered question that gets to the heart of understanding infection: what are the microbial growth rates?

The Phosphate Binder Ferric Citrate Alters the Gut Microbiome in Rats with Chronic Kidney Disease.

In chronic kidney disease (CKD), the gut microbiome is altered and bacterial-derived uremic toxins promote systemic inflammation and cardiovascular disease. Ferric citrate complex is a dietary phosphate binder prescribed for patients with end-stage kidney disease to treat hyperphosphatemia and secondary hyperparathyroidism. Iron is an essential nutrient in both microbes and mammals. This study was undertaken to test the hypothesis that the large iron load administered with ferric citrate in CKD may significantly change the gut microbiome. Male Sprague-Dawley rats underwent 5/6 nephrectomy to induce CKD. Normal control and CKD rats were randomized to regular chow or a 4% ferric citrate diet for 6 weeks. Fecal and cecal microbial DNA was analyzed via 16S ribosomal RNA gene sequencing on the Illumina MiSeq system. CKD rats had lower abundances of Firmicutes and Lactobacillus compared with normal rats and had lower overall gut microbial diversity. CKD rats treated with ferric citrate had improved hemoglobin and creatinine clearance and amelioration of hyperphosphatemia and hypertension. Ferric citrate treatment increased bacterial diversity in CKD rats almost to levels observed in control rats. The tryptophanase-possessing families Verrucomicrobia, Clostridiaceae, and Enterobacteriaceae were increased by ferric citrate treatment. The uremic toxins indoxyl sulfate and p-cresyl sulfate were not increased with ferric citrate treatment. Verrucomicrobia was largely represented by Akkermansia muciniphila, which has important roles in mucin degradation and gut barrier integrity. In summary, ferric citrate therapy in CKD rats was associated with significant changes in the gut microbiome and beneficial kidney and blood pressure parameters.

Precision synbiotics increase gut microbiome diversity and improve gastrointestinal symptoms in a pilot open-label study for autism spectrum disorder.

The efficacy of prebiotics and probiotics (synbiotics when combined) to improve symptoms associated with autism spectrum disorder (ASD) has shown considerable inter-study variation, likely due to the complex, heterogeneous nature of the disorder and its associated behavioral, developmental, and gastrointestinal symptoms. Here, we present a precision synbiotic supplementation study in 296 children and adults diagnosed with ASD versus 123 age-matched neurotypical controls. One hundred seventy ASD participants completed the study. Baseline and post-synbiotic assessment of ASD and gastrointestinal (GI) symptoms and deep metagenomic sequencing were performed. Within the ASD cohort, there were significant differences in microbes between subpopulations based on the social responsiveness scale (SRS2) survey (Prevotella spp., Bacteroides, Fusicatenibacter, and others) and gluten and dairy-free diets (Bifidobacterium spp., Lactococcus, Streptococcus spp., and others). At the baseline, the ASD cohort maintained a lower taxonomic alpha diversity and significant differences in taxonomic composition, metabolic pathways, and gene families, with a greater proportion of potential pathogens, including Shigella, Klebsiella, and Clostridium, and lower proportions of beneficial microbes, including Faecalibacterium compared to controls. Following the 3-month synbiotic supplementation, the ASD cohort showed increased taxonomic alpha diversity, shifts in taxonomy and metabolic pathway potential, and improvements in some ASD-related symptoms, including a significant reduction in GI discomfort and overall improved language, comprehension, cognition, thinking, and speech. However, the open-label study design may include some placebo effects. In summary, we found that precision synbiotics modulated the gut microbiome and could be used as supplementation to improve gastrointestinal and ASD-related symptoms. IMPORTANCE: Autism spectrum disorder (ASD) is prevalent in 1 out of 36 children in the United States and contributes to health, financial, and psychological burdens. Attempts to identify a gut microbiome signature of ASD have produced varied results. The limited pre-clinical and clinical population sizes have hampered the success of these trials. To understand the microbiome associated with ASD, we employed whole metagenomic shotgun sequencing to classify microbial composition and genetic functional potential. Despite being one of the most extensive ASD post-synbiotic assessment studies, the results highlight the complexity of performing such a case-control supplementation study in this population and the potential for a future therapeutic approach in ASD.

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host's microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples. Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Alterations in Gut Microbiome Composition and Function in Irritable Bowel Syndrome and Increased Probiotic Abundance with Daily Supplementation.

Irritable bowel syndrome (IBS) is characterized by abdominal discomfort and irregular bowel movements and stool consistency. As such, the gut microbiome has been posited as being influential for the syndrome. However, identifying microbial features associated with IBS symptom heterogeneity is difficult without large cohorts. Our aim was to identify microbial features associated with IBS and IBS subtypes compared to healthy controls and to determine if a synbiotic supplementation intervention could decrease the proportion of those microbial features. Stool samples from 490 individuals with IBS (including all dominant subtypes) and 122 individuals without IBS were analyzed with metagenomic sequencing. One hundred thirty-four IBS subjects were followed over time while receiving daily synbiotic supplementation, the composition of which varied between participants. IBS participants had significantly lower alpha diversity, an enrichment in Gram-negative bacteria, and a reduction in pathways associated with short-chain fatty acid and vitamin synthesis. Shigella species were significantly associated with IBS, while Eubacterium rectale and Faecalibacterium prausnitzii were associated with healthy controls. Random forest identified unique and overlapping microbial features associated with each IBS subtype. Longitudinal assessment of 134 IBS subjects receiving synbiotic supplements demonstrated a significant difference in microbial features and an increase in probiotic abundance across time. We identified microbial features that differentiate healthy and IBS subtypes. Synbiotic supplementation in IBS subjects did not result in alpha diversity change in the microbiome but did demonstrate changes in microbial features. Future work is needed to determine if the observed microbiome changes are associated with IBS symptom improvement. IMPORTANCE An estimated 35 million people in the United States and 11.5% of the population globally are affected by IBS. Immunity, genetics, environment, diet, small intestinal bacterial overgrowth (SIBO), and the gut microbiome are all factors that contribute to the onset or triggers of IBS. With strong supporting evidence that the gut microbiome may influence symptoms associated with IBS, elucidating the important microbes that contribute to the symptoms and severity is important to make decisions for targeted treatment. As probiotics have become more common in treating IBS symptoms, identifying effective probiotics may help inform future studies and treatment.

Lumacaftor/ivacaftor changes the lung microbiome and metabolome in cystic fibrosis patients.

RATIONALE: Targeted cystic fibrosis (CF) therapy with lumacaftor/ivacaftor partly restores chloride channel function and improves epithelial fluid transport in the airways. Consequently, changes may occur in the microbiome, which is adapted to CF lungs. OBJECTIVES: To investigate the effects of lumacaftor/ivacaftor on respiratory microbial composition and microbial metabolic activity by repeatedly sampling the lower respiratory tract. METHODS: This was a single-centre longitudinal observational cohort study in adult CF patients with a homozygous Phe508del mutation. Lung function measurements and microbial cultures of sputum were performed as part of routine care. An oral and nasal wash, and a breath sample, were collected before and every 3 months after starting therapy, for up to 12 months. RESULTS: Twenty patients were included in this study. Amplicon 16S RNA and metagenomics sequencing revealed that Pseudomonas aeruginosa was most abundant in sputum and seemed to decrease after 6 months of treatment, although this did not reach statistical significance after correction for multiple testing. Two types of untargeted metabolomics analyses in sputum showed a change in metabolic composition between 3 and 9 months that almost returned to baseline levels after 12 months of treatment. The volatile metabolic composition of breath was significantly different after 3 months and remained different from baseline until 12 months follow-up. CONCLUSIONS: After starting CF transmembrane conductance regulator (CFTR) modulating treatment in CF patients with a homozygous Phe508del mutation, a temporary and moderate change in the lung microbiome is observed, which is mainly characterised by a reduction in the relative abundance of Pseudomonas aeruginosa.

Thriving Under Stress: Pseudomonas aeruginosa Outcompetes the Background Polymicrobial Community Under Treatment Conditions in a Novel Chronic Wound Model.

In vitro infection models are important for studying the effects of antimicrobials on microbial growth and metabolism. However, many models lack important biological components that resemble the polymicrobial nature of chronic wounds or infections. In this study, we developed a perfused meat model that supports the growth of the human pathogen Pseudomonas aeruginosa in a native meat microbial background to investigate the impact of antibiotics and hydrogen peroxide on polymicrobial community growth and metabolism. P. aeruginosa plays an important role as an etiological agent involved in chronic infections and is a common opportunistic pathogen. Chemical stressors in the form of hydrogen peroxide, carbenicillin, and gentamicin were perfused through the meat with polymicrobial growth on the surface. The relative abundances of P. aeruginosa and the background microbial community were analyzed by cell viability assays, and metabolic changes of the entire community in response to different antimicrobial treatments were characterized by GC-MS analysis of volatile organic compounds. The meat background community was characterized by amplicon sequencing. Relative densities of P. aeruginosa and background microbiota were similar under control conditions. Antimicrobial stressors, even at sub-inhibitory, physiologically relevant concentrations, spurred P. aeruginosa dominance of the meat surface community. Volatile metabolite ion intensity levels showed that antibacterial treatments drive changes in microbial metabolism. The abundance of the P. aeruginosa-derived metabolite, acetophenone, remained stable with treatment, whereas the relative abundances of 2-butanone, 2-nonanone, and 2-aminoacetophenone changed in response to treatment, suggesting these could serve as biomarkers of infection. Our model recapitulates some of the physiological conditions of chronic wounds and facilitates high throughput experiments without the high cost of in vivo models. Expanded use of this perfusion model will contribute to the understanding of polymicrobial growth and metabolism in the context of chronic wounds and infections.

Longitudinal Associations of the Cystic Fibrosis Airway Microbiome and Volatile Metabolites: A Case Study.

The identification of 16S rDNA biomarkers from respiratory samples to describe the continuum of clinical disease states within persons having cystic fibrosis (CF) has remained elusive. We sought to combine 16S, metagenomics, and metabolomics data to describe multiple transitions between clinical disease states in 14 samples collected over a 12-month period in a single person with CF. We hypothesized that each clinical disease state would have a unique combination of bacterial genera and volatile metabolites as a potential signature that could be utilized as a biomarker of clinical disease state. Taxonomy identified by 16S sequencing corroborated clinical culture results, with the majority of the 109 PCR amplicons belonging to the bacteria grown in clinical cultures (Escherichia coli and Staphylococcus aureus). While alpha diversity measures fluctuated across disease states, no significant trends were present. Principle coordinates analysis showed that treatment samples trended toward a different community composition than baseline and exacerbation samples. This was driven by the phylum Bacteroidetes (less abundant in treatment, log2 fold difference -3.29, p = 0.015) and the genus Stenotrophomonas (more abundant in treatment, log2 fold difference 6.26, p = 0.003). Across all sputum samples, 466 distinct volatile metabolites were identified with total intensity varying across clinical disease state. Baseline and exacerbation samples were rather uniform in chemical composition and similar to one another, while treatment samples were highly variable and differed from the other two disease states. When utilizing a combination of the microbiome and metabolome data, we observed associations between samples dominated Staphylococcus and Escherichia and higher relative abundances of alcohols, while samples dominated by Achromobacter correlated with a metabolomics shift toward more oxidized volatiles. However, the microbiome and metabolome data were not tightly correlated; examining both the metagenomics and metabolomics allows for more context to examine changes across clinical disease states. In our study, combining the sputum microbiome and metabolome data revealed stability in the sputum composition through the first exacerbation and treatment episode, and into the second exacerbation. However, the second treatment ushered in a prolonged period of instability, which after three additional exacerbations and treatments culminated in a new lung microbiome and metabolome.

Fermentation products in the cystic fibrosis airways induce aggregation and dormancy-associated expression profiles in a CF clinical isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a well-known dominant opportunistic pathogen in cystic fibrosis (CF) with a wide range of metabolic capacities. However, P. aeruginosa does not colonize the airways alone, and benefits from the metabolic products of neighboring cells-especially volatile molecules that can travel between different parts of the airways easily. Here, we present a study that investigates the metabolic, gene expression profiles and phenotypic responses of a P. aeruginosa clinical isolate to fermentation products lactic acid and 2,3-butanediol, metabolites that are produced by facultative anaerobic members of the CF polymicrobial community and potential biomarkers of disease progression. Although previous studies have successfully investigated the metabolic and transcriptional profiles of P. aeruginosa, most have used common lab reference strains that may differ in important ways from clinical isolates. Using transcriptomics and metabolomics with gas chromatography time of flight mass spectrometry, we observe that fermentation products induce pyocyanin production along with the expression of genes involved in P. aeruginosa amino acid utilization, dormancy and aggregative or biofilm modes of growth. These findings have important implications for how interactions within the diverse CF microbial community influence microbial physiology, with potential clinical consequences.

Stable isotope profiles reveal active production of VOCs from human-associated microbes.

Volatile organic compounds (VOCs) measured from exhaled breath have great promise for the diagnosis of bacterial infections. However, determining human or microbial origin of VOCs detected in breath remains a great challenge. For example, the microbial fermentation product 2,3-butanedione was recently found in the breath of Cystic Fibrosis (CF) patients; parallel culture-independent metagenomic sequencing of the same samples revealed that Streptococcus and Rothia spp. have the genetic capacity to produce 2,3-butanedione. To investigate whether the genetic capacity found in metagenomes translates to bacterial production of a VOC of interest such as 2,3-butanedione, we fed stable isotopes to three bacterial strains isolated from patients: two gram-positive bacteria, Rothia mucilaginosa and Streptococcus salivarius, and a dominant opportunistic gram-negative pathogen, Pseudomonas aeruginosa. Culture headspaces were collected and analyzed using a gas chromatographic system to quantify the abundance of VOCs of interest; mass spectroscopy was used to determine whether the stable isotope label had been incorporated. Our results show that R. mucilaginosa and S. salivarius consumed D-Glucose-13C6 to produce labeled 2,3-butanedione. R. mucilaginosa and S. salivarius also produced labeled acetaldehyde and ethanol when grown with 2H2O. Additionally, we find that P. aeruginosa growth and dimethyl sulfide production are increased when exposed to lactic acid in culture. These results highlight the importance VOCs produced by P. aeruginosa, R. mucilaginosa, and S. salivarius as nutrients and signals in microbial communities, and as potential biomarkers in a CF infection.

Diverse genetic architectures lead to the same cryptic phenotype in a yeast cross.

Cryptic genetic variants that do not typically influence traits can interact epistatically with each other and mutations to cause unexpected phenotypes. To improve understanding of the genetic architectures and molecular mechanisms that underlie these interactions, we comprehensively dissected the genetic bases of 17 independent instances of the same cryptic colony phenotype in a yeast cross. In eight cases, the phenotype resulted from a genetic interaction between a de novo mutation and one or more cryptic variants. The number and identities of detected cryptic variants depended on the mutated gene. In the nine remaining cases, the phenotype arose without a de novo mutation due to two different classes of higher-order genetic interactions that only involve cryptic variants. Our results may be relevant to other species and disease, as most of the mutations and cryptic variants identified in our study reside in components of a partially conserved and oncogenic signalling pathway.

Regulatory Rewiring in a Cross Causes Extensive Genetic Heterogeneity.

Genetic heterogeneity occurs when individuals express similar phenotypes as a result of different underlying mechanisms. Although such heterogeneity is known to be a potential source of unexplained heritability in genetic mapping studies, its prevalence and molecular basis are not fully understood. Here we show that substantial genetic heterogeneity underlies a model phenotype--the ability to grow invasively--in a cross of two Saccharomyces cerevisiae strains. The heterogeneous basis of this trait across genotypes and environments makes it difficult to detect causal loci with standard genetic mapping techniques. However, using selective genotyping in the original cross, as well as in targeted backcrosses, we detected four loci that contribute to differences in the ability to grow invasively. Identification of causal genes at these loci suggests that they act by changing the underlying regulatory architecture of invasion. We verified this point by deleting many of the known transcriptional activators of invasion, as well as the gene encoding the cell surface protein Flo11 from five relevant segregants and showing that these individuals differ in the genes they require for invasion. Our work illustrates the extensive genetic heterogeneity that can underlie a trait and suggests that regulatory rewiring is a basic mechanism that gives rise to this heterogeneity.